Minor groove binder (MGB) phosphoramidites are new tools that allow adding the MGB moiety dihydropyrroloindole-carboxylate (CDPI3) to the 3’- or 5’-end of oligonucleotides.
MGB phosphoramidites can be used for the synthesis of modified oligonucleotides that contain the MGB moiety and have increased duplex stability useful in PCR, DNA array and antisense applications.
Adding bridged nucleic acids to selected positions within the oligonucleotide sequence will increase the stability of the final DNA duplex even more.
The structures of the two commercially CDPI3 MGB phosphoramidites are shown below.
Figure 1: 5’-CDPI3, MGB Phosphoramidite. This phosporamidite allows adding the MGB moiety to the 5’-end of an oligonucleotide during solid phase oligonucleotide synthesis.
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Figure 2: CDPI3, MGB CPG. This phosporamidite allows adding the MGB moiety to the 3’-end of an oligonucleotide during solid phase oligonucleotide synthesis.
Kutyavin et al. in 2000 designed and synthesized a fluorogenic DNA probes conjugated to a minor groove binder moiety. These new MGB probes were more specific than standard DNA probes. The probes stabilized A/T rich duplexes more than G/C duplexes.
Figure 3: DNA duplex formed with MGB-oligonucleotide probe.
If a MGB-oligonucleotide probe is designed using FRET pairs such as a reported dye, for example, fluorescein, on the 5’-end, and an internal quencher, for example, TAMRA, upstream of the MGB moiety, the probe can be used for real-time measurements in PCR.
The hybridized probe is cleaved during the primer extension step by the 5’-exonuclease activity of Taq polymerase which results in the release of a fluorescent signal. The fluorescent signal can be detected by any quantitative PCR instrument that combines thermal cycler functions with a fluorimeter.
Several types of MGB probes have been developed and tested in recent years. These are:
(1) 5’-F-3’-Q MGB TAQMAN probes
(2) 3’-F-5’ Q FRET MGB probes
(3) 3’-Q-5’-F FRET MGB probes
(4) miRNA inhibitor probes
These new types of probes increase the repertoire of molecular tools useful for diagnostics and gene expression modulation.
We at Biosynthesis Inc. have extensive experience in DNA and RNA synthesis as well as in probe design.
Reference
MGB-probes: http://www.glenresearch.com/GlenReports/GR29-11.html
Kutyavin, I. V., Afonina, I. A., Mills, A., Gorn, V. V., Lukhtanov, E. A., Belousov, E. S., … Hedgpeth, J. (2000). 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Research, 28(2), 655–661. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC102528/